Cell cultivation method and cell culture

ABSTRACT

Provided is a cell cultivation method in which the cell is cultured using a peptide hydrogel as a scaffold, for carrying out high-dimensional culture of a cell such as porcine hepatocyte, human hepatocyte, porcine pancreatic islet or human pancreatic islet for a long period under conditions where cell survival, cell morphology and cell functions are maintained. Also provided are a cell culture including a cell and a peptide hydrogel obtained by the above-described cultivation method, a bioreactor including the cell culture, and a cell preparation including the cell culture.

TECHNICAL FIELD

The present invention relates to a cell cultivation method and a cellculture.

BACKGROUND ART

Internal organs to be subjected to transplantation include, for example,liver. Liver is the largest parenchymatous organ in a human body. It hasvarious functions such as bilirubin metabolism, drug metabolism, andblood coagulation factor production, typically including metabolisms ofcarbohydrates, proteins and lipids, playing a very important role in anorganism.

Thus, severe hepatic failure is very dangerous for the life of a patienteven if it is temporary. On the other hand, if a liver function can besubstituted for about one week, due to highly regenerative ability ofliver, a patient with liver damage caused by fulminant liver failurerecovers. For such a serious liver disease, hepatic transplantation isthe most effective therapy. However, not all the patient can receive itsfavor due to serious donor shortage. Under the current actual conditionsin Japan, although the life-saving rate has been raised to some extentby a combination of continuous filtration dialysis and plasma exchangeas the means for temporarily substituting a liver function, the rate isstill insufficient (see, Abe, et al., “Study of plasma exchange forliver failure: beneficial and harmful effects.”, The Apher Dial, 2004,8, p. 180-184), and the establishment of more effective treatmentmethods have been eagerly desired.

Under such circumstances, therapeutic artificial livers are highlyexpected, especially, development of bioartificial livers filled withcells in order to utilize a metabolic ability and a protein synthesisability of living cells is attracting attention. A bioartificial liveris a module prepared by incorporating and fixing hepatocytes incarriers, and it can be said as an artificial liver device simulatingliver in human body. The blood in a subject is introduced in the deviceand the removal of harmful substances in the blood and the feed ofbiologically active substances such as coagulation factor derived fromthe liver cells can be carried out by utilizing the metabolizing abilityof hepatocytes.

In one example of the experiments of human hepatocytes culturing by thepresent inventors, the donor was a white male (56 years old) who hadexperienced head trauma by a traffic accident and the cause of his deathwas subarachnoid hemorrhage. The transport time from US was about 39hours. Hepatocytes were separated by normograde perfusion using acollagenase and then, cold-preserved in William's Medium E, andair-transported from Chiba prefecture to Okayama prefecture. Thehepatocytes were cultured in culture medium comprising mainly William'sMedium E to which deleted form hepatocyte growth factor (dHGF) was addedat various concentrations of 0 ng/ml for group A), 10 ng/ml for groupB), 100 ng/ml for group C) and 1000 ng/ml for group D), and the effectsof dHGF were assessed based on a growth ability by the MIT assay;metabolic abilities of ammonia, lidocaine and diazepam, and an albuminproduction ability.

In the dHGF-added groups, cell growth was significantly better.Metabolic abilities of ammonia, lidocaine and diazepam and an albuminproduction ability were significantly better in the groups B) and C),while in the group D), 1000 ng/ml dHGF addition, a drug metabolicability and an ammonia production ability per given cell number lowered.Visual observation also showed that a cobblestone morphology ofhepatocytes was maintained well in the groups B) and C).

In Europe and the United States, hepatocytes are separated from theliver unsuitable for transplantation, and adopted clinically tohepatocyte transplantation and bioartificial livers. However, in Japan,the liver unsuitable for transplantation is prescribed as incinerationand thus, it cannot be used for the bioartificial livers. Therefore, adonor liver judged to be unsuitable for transplantation (because ofreasons such as fatty liver and intense fibrosis) in the United Stateswas obtained from National Disease Research Interchange (NDR1) via HUMAN& ANIMAL BRIDGING RESEARCH ORGANIZATION laboratory (Ichikawa city, Chibaprefecture, responsible person: Dr. Satoshi Suzuki) in the form of liverblock (130 g) which was then separated into liver cells, and afunctional cultivation method thereof was investigated. It is extremelyimportant to develop a cultivation method aiming at functionalmaintenance of human hepatocytes separated from such donor liverunsuitable for transplantation that is only usable resource.

As a countermeasure for the problem wherein use of healthy humanhepatocytes is impossible, there have been trials of induction intohepatocytes from human peripheral blood stem cells, myeloid stem cellsand liver precursor cells. These cells, however, show poor growthability and thus, it is not realistic to obtain sufficient number ofcells necessary for application to bioartificial livers (at least onebillion). Therefore, in Europe and China, clinical trials ofbioartificial livers using porcine hepatocytes have been carried out inhumans (see, van de Kerkhove, et al., “Phase I clinical trial with theAMC-bioartificial liver.”, Academic Medical Center Int J Artif Organs,2002, 25, p. 950-959, Donini, et al., “Temporary neurologicalimprovement in a patient with acute or chronic liver failure treatedwith a bioartificial liver device”, Am J Gastroentrol, 2000, 95, p.1102-1104, Mazariegos, et al., “Safety observations in phase I clinicalevaluation of the Excorp Medical Bioartificial Liver Support Systemafter the first four patients.”, ASAIO J, 2001, 47, p. 471-475, Ding, etal., “The development of a new bioartificial liver and its applicationin 12 acute liver failure patients.”, World J Gastroenterol, 2003, 9, p.829-832, Demetriou, et al., “Prospective, randomized, multicenter,controlled trial of a bioartificial liver in treating acute liverfailure.”, Ann Surgery, 2004, 239, p. 660-670, Mundt, “A method toassess biochemical activity of liver cells during clinical applicationof extracorporeal hybrid liver support.”, Int J Artif Organs, 2002, 25,p. 542-548, Morsiani, et al., “Early experiences with a porcinehepatocyte-based bioartificial liver in acute hepatic failurepatients.”, Int J Artif Organs, 2002, 25, p. 192-202, Xue, et al., “TECAhybrid artificial liver support system in treatment of acute liverfailure.”, World J Gastroenterol, 2001, 7, p. 826-829).

It is expected that cell or tissue culture technologies can be adoptedindustrially for regenerative medicine, cell preparation, usefulsubstance production (bioreactor), investigation and research intofunction of tissue, organ and internal organ, screening of new drugs,animal experiment substitute methods for evaluating influences ofendocrine disrupting chemicals, and cell chips, typically including celltransplantation and bioartificial organs.

Conventionally, as a method for culturing animal cells havingadhesiveness, a two-dimensional cultivation method, that is, a so-calledmonolayer cultivation method has been generally used, in which asubstrate such as a culture dish made of polystyrene or glass is usedand the surface thereof is coated with a living body-derived factor, ortreated chemically or physicochemically, and cells are adhered to andspread on the surface. For example, if cells are cultured on apolystyrene dish coated with collagen, an animal-derived intercellularmatrix component, or on a polystyrene dish having a surfacehydrophilized by plasma treatment, the cells adhere to and spread on thesurface, thereby taking a cell morphology in which cytoplasm is spreadin flat form.

On the other hand, cells isolated from tissue and internal organs oforganisms, so-called primary cells, often maintain properties andfunctions of tissue and internal organs from which the cells areoriginated and thus, these cells have a great deal of potential inindustrial application. However, it is known that in the monolayercultivation method, properties and functions of various cells will belost in several days or several weeks, in most cases. Particularly, inthe case of primary hepatocytes which are well-differentiated and havevarious complicated functions among primary cells, properties andfunctions thereof tend to be lost quickly in the monolayer culture. Forexample, it is known that if hepatocytes isolated from rat liver aremonolayer-cultured, important functions of liver, that is, a proteinsynthesis function, a detoxification function and a drug metabolicfunction are lowered or lost within several days from initiation ofculture. It is hypothesized that in a monolayer cultivation method,cells have cytoplasm in the form of flat two-dimensional state and thus,mechanisms originally possessed by cells in a living body, such as anintracellular structure, polarity, and information exchange due tobonding with adjacent cells, are lowered and lost, causing lowering andloss of properties and functions originally possessed by cells (see,Japanese Unexamined Patent Publication No. 128660/2001).

In order to avoid such lowering and loss of the properties and functionsoriginally possessed by cells, a so-called “three-dimensionalcultivation method” has drawn attention in which cells are mutuallyassembled to construct a three-dimensional structure similar to livingtissue. Scaffolds in such a three-dimensional cultivation method areroughly classified into two types. One is an animal-derivedintercellular matrix component, and another is a synthetic polymer.Examples of the animal-derived intercellular matrix component includecollagen gel, laminin, and animal basement membrane-derived component(trade name: Matrigel, available from Becton Dickinson and Company(constituents: laminin 56%, collagen IV 31% and entactin 8%)). It isreported that when, for example, rat hepatocytes are cultured withMatrigel, spheroid is formed (see, Bissell, et al., “Transcriptionalregulation of the albumin gene in cultured rat hepatocytes. Role ofbasement-membrane matrix.”, Mol Biol Med, 1990, 7, p. 187-197). Examplesof the synthetic polymer include polyglucosic acid and poly L-lacticacid. It is reported that when, for example, rat hepatocytes arecultured with polyglucosic acid, spheroid is formed (see, Fiegel, etal., “Influence of flow conditions and matrix coatings on growth anddifferentiation of three-dimensionally cultured rat hepatocytes.”,Tissue Eng, 2004, 10, p. 165-174).

Since cells cultured by the three-dimensional cultivation method arecapable of maintaining properties and functions originally possessed bycells at higher level for a longer period of time as compared with cellscultured by a two-dimensional cultivation method, as described above, itis anticipated that the cells cultured by a three-dimensionalcultivation method can be highly effective means for industrialapplications such as bioartificial organs, regeneration medicine, cellpreparation, useful substance production (bioreactor), investigation andresearch of function of tissue, organ and internal organ, screening ofnew drugs, animal experiment substitute methods for evaluatinginfluences of endocrine disrupting chemicals, and cell chips.

DISCLOSURE OF INVENTION

However, the above-described scaffolds in the three-dimensionalcultivation method had the following issues. That is, when ananimal-derived intercellular matrix component is used as a scaffold,reproducibility is poor due to a large variability between lots.Furthermore, there is a possibility of contamination with unknownfactors such as unknown viruses, and a fear of infection in clinicaluse. When a synthetic polymer is used as a scaffold, the fiber diameteris as large as 10 to 50 μm and thus, the environment is substantiallythe same as in flat culture when taking the size of cells (5 to 20 μm)into consideration. Additionally, since inter-fiber size (pore size) isas large as 10 to 200 μm, there is a problem that intercellular matrixcomponents produced by cells do not remain in the scaffold, and suspendinto culture medium. Furthermore, there is a problem that the componentsremain in a body as extraneous materials in case of transplantation,because of low biodegradability.

In view of the above-described issues, an objective of the presentinvention is to provide a technology in order to performhigh-dimensional culture for a long period of time for cells such asporcine hepatocyte, human hepatocyte, porcine pancreatic islet and humanpancreatic islet, under conditions where cell survival, cell morphologyand cell functions are maintained.

As a result of intense study for achieving the above-describedobjective, it has been found that the above-described objective can beattained by culturing a cell using a peptide hydrogel having the samefiber size and pore size as those of natural intercellular matrices, andhaving biocompatibility and biodegradability, leading to completion ofthe present invention as a scaffold.

That is, the present invention relates to a method for culturing a cellwherein the cell is cultured using a peptide hydrogel as a scaffold.

In a preferable embodiment of the present invention, the cell iscultured further using an insert.

In a further preferable embodiment of the present invention, theabove-described cell is at least one member selected from the groupconsisting of porcine hepatocyte, human hepatocyte, porcine pancreaticislet and human pancreatic islet.

The present invention also relates to a cell culture comprising a celland a peptide hydrogel obtained by the above-described cultivationmethod.

In a preferable embodiment of the present invention, cells in theabove-described culture constitute a cell culture having a spheroidmorphology.

In a further preferable embodiment of the present invention, cells inthe above-described culture are hepatocytes having formation of a celladhesion apparatus and/or bile canaliculi.

In a further preferable embodiment of the present invention, cells inthe above-described culture constitute a pancreatic islet showing atleast 12 points in the sum of evaluation values regarding pancreaticislet morphological criteria: shape, border shape, cell integrity andcell diameter and in which the ratio of insulin secretion in low glucoseconcentration to that in high glucose concentration to glucosestimulation is at least 1.5-fold.

The present invention also relates to a bioreactor including a cellculture obtained by culturing hepatocyte using a peptide hydrogel as ascaffold.

In a preferable embodiment of the present invention, the above-describedbioreactor metabolizes ammonia, diazepam or lidocaine.

The present invention also relates to a bioreactor including a cellculture obtained by culturing pancreatic islet using a peptide hydrogelas a scaffold.

In a preferable embodiment of the present invention, the above-describedbioreactor produces insulin.

The present invention further relates to a cell preparation including acell culture obtained by culturing cells using a peptide hydrogel as ascaffold.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1( a) is a schematic drawing showing one example of a culturevessel used in a cultivation method of the present invention using apeptide hydrogel as a scaffold. Numerical reference 1 shows a culturedish, and numerical reference 2 shows an insert. A filter 3 is providedat the bottom of the insert 2. The insert 2 is inserted in the culturedish 1. A peptide hydrogel is spread on the filter 3 of the insert 2,and a cell is placed thereon and cultured. FIG. 1( b) is across-sectional drawing showing one embodiment of a cultivation methodof the present invention using the culture vessel shown in FIG. 1( a).Numerical reference 4 shows a cell. The peptide hydrogel is notillustrated.

FIG. 2( a) is a phase-contrast microscopy image showing cultureconditions of porcine hepatocytes cultured by a cultivation method ofthe present invention using a peptide hydrogel as a scaffold(Photographs 1, 2 and 3) and porcine hepatocytes cultured by aconventional cultivation method using collagen as a scaffold(Photographs 4, 5 and 6).

FIG. 2( b) is a scanning electron microscopy image showing cultureconditions of porcine hepatocytes cultured by a cultivation method ofthe present invention using a peptide hydrogel as a scaffold(Photographs 1, 2 and 3) and porcine hepatocytes cultured by aconventional cultivation method using collagen as a scaffold(Photographs 4, 5 and 6).

FIG. 2( c) is a transmission electron microscopy image showing thecross-section of porcine hepatocyte cultured using an insert in acultivation method of the present invention using a peptide hydrogel asa scaffold, showing that the porcine hepatocyte constitutes ahigh-dimensional culture. Between cells, a cell adhesion apparatus andbile canaliculi are formed.

FIG. 2( d) is a drawing for explaining a cell adhesion apparatus andbile canaliculi in FIG. 2( c). Numerical reference 5 shows a celladhesion apparatus, and numerical reference 6 shows a bile canaliculi.Numerical reference 7 shows an interface between cells.

FIG. 3 is a phase-contrast microscopy image showing a viability statusof porcine hepatocyte cultured by a cultivation method of the presentinvention using a peptide hydrogel as a scaffold (Photographs 1, 2 and3) and porcine hepatocyte cultured by a conventional cultivation methodusing collagen as a scaffold (Photographs 4, 5 and 6).

FIG. 4( a) is a graph comparing metabolic abilities of ammonia ofporcine hepatocyte cultured by a cultivation method of the presentinvention using a peptide hydrogel as a scaffold (group A) and porcinehepatocyte cultured by a conventional cultivation method using collagenas a scaffold (group B).

FIG. 4( b) is a graph comparing metabolic abilities of ammonia ofporcine hepatocyte cultured by a cultivation method of the presentinvention using a peptide hydrogel as a scaffold (group A), porcinehepatocyte cultured by a conventional cultivation method using collagenas a scaffold (group B) and porcine hepatocyte cultured by a cultivationmethod using Matrigel as a scaffold (group C).

FIG. 4( c) is a graph comparing metabolic abilities of ammonia ofporcine hepatocyte cultured using an insert, in a cultivation method ofthe present invention using a peptide hydrogel as a scaffold (group A),a conventional cultivation method using collagen as a scaffold (groupB), a cultivation method using Matrigel as a scaffold (group C) and acultivation method using collagen sandwich as a scaffold (group D),respectively.

FIG. 5( a) is a graph comparing metabolic abilities of lidocaine inporcine hepatocyte cultured by a cultivation method of the presentinvention using a peptide hydrogel as a scaffold (group A) and porcinehepatocyte cultured by a conventional cultivation method using collagenas a scaffold (group B).

FIG. 5( b) is a graph comparing metabolic abilities of lidocaine inporcine hepatocyte cultured by a cultivation method of the presentinvention using a peptide hydrogel as a scaffold (group A), porcinehepatocyte cultured by a conventional cultivation method using collagenas a scaffold (group B) and porcine hepatocyte cultured by a cultivationmethod using Matrigel as a scaffold (group C).

FIG. 5( c) is a graph comparing metabolic abilities of lidocaine inporcine hepatocyte cultured using an insert in addition, in acultivation method of the present invention using a peptide hydrogel asa scaffold (group A), a conventional cultivation method using collagenas a scaffold (group B), a cultivation method using Matrigel as ascaffold (group C) and a cultivation method using collagen sandwich as ascaffold (group D), respectively.

FIG. 6( a) is a graph comparing metabolic abilities of diazepam inporcine hepatocyte cultured by a cultivation method of the presentinvention using a peptide hydrogel as a scaffold (group A) and porcinehepatocyte cultured by a conventional cultivation method using collagenas a scaffold (group B).

FIG. 6( b) is a graph comparing metabolic abilities of diazepam inporcine hepatocyte cultured by a cultivation method of the presentinvention using a peptide hydrogel as a scaffold (group A), porcinehepatocyte cultured by a conventional cultivation method using collagenas a scaffold (group B) and porcine hepatocyte cultured by a cultivationmethod using Matrigel as a scaffold (group C).

FIG. 6( c) is a graph comparing metabolic abilities of diazepam inporcine hepatocyte cultured by a cultivation method of the presentinvention using a peptide hydrogel as a scaffold (group A), aconventional cultivation method using collagen as a scaffold (group B),a cultivation method using Matrigel as a scaffold (group C) and acultivation method using collagen sandwich as a scaffold (group D), aswell as each cultivation methods with insert, respectively.

FIG. 7( a) is a phase-contrast microscopy image showing cultureconditions of human hepatocyte cultured by a cultivation method of thepresent invention using a peptide hydrogel as a scaffold (Photographs 1,2 and 3) and human hepatocyte cultured by a conventional cultivationmethod using collagen as a scaffold (Photographs 4, 5 and 6).

FIG. 7( b) is a scanning electron microscopy image showing cultureconditions of human hepatocyte cultured by a cultivation method of thepresent invention using a peptide hydrogel as a scaffold (Photographs 1,2 and 3) and human hepatocyte cultured by a conventional cultivationmethod using collagen as a scaffold (Photographs 4, 5 and 6).

FIG. 8( a) is a graph comparing metabolic abilities of ammonia in humanhepatocyte cultured by a cultivation method of the present inventionusing a peptide hydrogel as a scaffold (group A) and human hepatocytecultured by a conventional cultivation method using collagen as ascaffold (group B).

FIG. 8( b) is a graph comparing metabolic abilities of ammonia in humanhepatocyte cultured by a cultivation method of the present inventionusing a peptide hydrogel as a scaffold (group A), human hepatocytecultured by a conventional cultivation method using collagen as ascaffold (group B) and human hepatocyte cultured by a cultivation methodusing Matrigel as a scaffold (group C).

FIG. 9 is a scanning electron microscopy image showing cultureconditions of porcine pancreatic islet cultured by a cultivation methodof the present invention using a peptide hydrogel as a scaffold (groupA) and porcine pancreatic islet cultured by a conventional cultivationmethod using collagen as a scaffold (group B).

FIG. 10( a) is a graph showing the insulin production ability of porcinepancreatic islet cultured by a cultivation method of the presentinvention using a peptide hydrogel as a scaffold (group A) and FIG. 10(b) is a graph showing the insulin production ability of porcinepancreatic islet cultured by a conventional cultivation method usingcollagen as a scaffold (group B).

FIG. 11 is a scanning electron microscopy image showing cultureconditions of porcine pancreatic islet cultured by a cultivation methodof the present invention using a peptide hydrogel as a scaffold (groupA) and porcine pancreatic islet cultured by a conventional cultivationmethod using collagen as a scaffold (group B).

FIG. 12( a) is a graph showing the insulin production ability of humanpancreatic islet cultured by a cultivation method of the presentinvention using a peptide hydrogel as a scaffold (group A) and FIG. 12(b) is a graph showing the insulin production ability of human pancreaticislet cultured by a conventional cultivation method using collagen as ascaffold (group B).

FIG. 13 is a drawing showing manufacturing processes in turn using oneembodiment of a bioreactor of the present invention as an example. (a)is a schematic drawing showing the condition of hollow fibers 12arranged on nonwoven fabric 11 equipped with lining 10. Here, thenonwoven fabric 11 equipped with lining 10 has a slit 13. (b) is aschematic drawing showing a process of winding the material in a form ofsheet shown in (a) in the form of roll. (c) is an X-X line cross-sectionenlarged drawing of (b). (d) is a schematic drawing showing a bioreactor16 which can be utilized in a bioartificial liver having a roll composedof the hollow fibers 12 and the nonwoven fabric 11 incorporated in acylindrical vessel 15 equipped with liquid leak prevention parts 14 onboth ends. The bioreactor 16 is equipped with a cell injection port 17and a discharge port 18 by which a cell sample can be collected, and theslit 13 is disposed so as to communicate with the cell injection port17.

BEST MODE FOR CARRYING OUT THE INVENTION

A cell used as subject matters in the present invention includes, forexample, hepatocyte of mammals such as porcine, monkey, anthropoid andhuman or pancreatic islet of mammals such as porcine, monkey, anthropoidand human. Among these, preferable is at least one member selected fromthe group consisting of porcine hepatocyte, human hepatocyte, porcinepancreatic islet and human pancreatic islet.

In the present invention, a self-assembling peptide hydrogel is adoptedas the scaffold to be used in performing high-dimensional culture ofcells such as porcine hepatocyte, human hepatocyte, porcine pancreaticislet and human pancreatic islet. As the peptide hydrogel, PuraMatrix ofSEQ ID NO:1 (Ac-(RADA)₄-CONH₂), EAK16 of SEQ ID NO:2(Ac-AEAEAKAKAEAEAKAK-CONH₂) and RAD 16 of SEQ ID NO: 3(Ac-RARADADARARADADA-CONH₂) are mentioned, and among them, PuraMatrix ofSEQ ID NO:1 commercially available from Three D Matrix Japan K. K. ispreferably mentioned.

Also, self-assembling peptides having an amino acid sequence and aminoacid residue number different from those of PuraMatrix of SEQ ID NO:1can be used as the scaffold. As preferable amino acid sequences,repetitions of X—N—Y—N(X represents a basic amino acid such as arginineand lysine, N represents a neutral amino acid such as alanine andglycine, Y represents an acidic amino acid such as aspartic acid andglutamic acid) are mentioned.

Since self-assembling peptides such as PuraMatrix of SEQ ID NO:1 havethe peptide sequences with no obvious physiologically active motif,there is no fear of deterioration of original cell functions. Aphysiologically active motif is correlated with control of manyintracellular phenomena such as transcription and thus, if aphysiologically active motif is present, proteins in cytoplasm and oncell surface are phosphorylated by an enzyme recognizing this motif. Ifa physiologically active motif is present in a scaffold, there is apossibility of suppression of transcription activating ability ofvarious functional proteins. For example, in a hepatocyte, side effectssuch as suppression of an albumin production ability and drug metabolicability can occur. In self-assembling peptides such as PuraMatrix of SEQID NO:1 having no physiologically active motif, such a fear does notexist. Therefore, self-assembling peptides such as PuraMatrix of SEQ IDNO:1 are scaffolds suitable for cell culture functioning only as aphysical cell foothold.

PuraMatrix of SEQ ID NO:1 is an oligo peptide containing 16 amino acidresidues (Ac-(RADA)₄-CONH₂) and having a length of about 5 nm, and itssolution is in liquid form when pH is lower than 5.0, when pH is changedto values of at least 5.0, self-assembly of peptide occurs, forming nanofibers having a diameter of about 10 nm. As a result, the peptidesolution will be gelated.

The above-described nano fibers have a diameter of 10 to 20 nm and apore size of 5 to 200 nm, on average. Since the range of these numericalvalue is approximately the same as those of collagen, a naturalintercellular matrix, self-assembling peptides are the suitable scaffoldfor cell culture.

PuraMatrix of SEQ ID NO:1 is an amphiphilic peptide having an amino acidsequence in which residues of positively charged arginine, negativelycharged aspartic acid and hydrophobic alanine repeat alternately, andself-assembly of peptide is ascribable to an ionic bond and ahydrophobic bond between the peptide molecules by amino acids comprisingthe peptide.

Self-assembly conditions of a self-assembling peptide includephysiological pH and a salt concentration. In particular, monovalentalkali metal ions are important, i.e., sodium ions and potassium ionspresent in large amount in an organism contribute to promotion ofgelation. Once gelled, the gel does not degrade even if usual proteindegeneration conditions, for example, high temperature, and degenerantssuch as acid, alkali, protease, urea and guanidine hydrochloride, areused.

This self-assembling peptide is capable of easily forming athree-dimensional porous scaffold, which is difficult to achieve withthe other technologies. The density and the average pore size of thenano fiber correlates to the concentration of the peptide solutionadopted. Depending on the concentration of an aqueous solution of aself-assembling peptide, the strength of the gel varies, and cultureconditions suitable for culturing cells can be obtained. It is thereforepossible to encapsulate cell within a three-dimensional environment, orto differentiate and grow cell successfully on the surface of a peptidehydrogel (see, Zhang, et al., Biomaterials, December; 16 (18): 1385-93,1995, Holmes, et al., Proc Natl Acad Sci USA. June 6; 97(12): 6728-33,2000, and Kisiday, et al., Proc Natl Acad Sci USA. July 23; 99(15):9996-10001, 2002).

Thus, a self-assembling peptide is capable of providing effectsequivalent to or more than those with animal-derived extracellularmatrices such as collagen, fibronectin and mouse sarcoma extracellularmatrix, in cell culture technologies. Furthermore, since aself-assembling peptide is produced through chemical synthesis, it doesnot include unknown components ascribable to animal-derivedextracellular matrices. This nature causes no fear of infectionsincluding BSE, also showing high safety for medical treatments.

A self-assembling peptide comprising natural amino acids is alsoexcellent in terms of its biocompatibility and degradability in theliving body. Thus, it is reported that when PuraMatrix of SEQ ID NO:1 isinjected into mouse myocardium, for example, cells penetrate into theinjected PuraMatrix of SEQ ID NO:1 and normal tissue is formed. Althoughthe degradation time varies depending on conditions such as injectionplace, fibers are degraded and discharged in about 2 to 8 weeks afterinjection.

Cell used as subject matters in the present invention includes cellcollected from human and animal or commercially available cell.Specifically mentioned are porcine hepatocyte, human hepatocyte, porcinepancreatic islet and human pancreatic islet.

Liver cells are used in the present invention can be obtained by amethod of excision from animal such as porcine and a method of usinghuman donor liver, as well as obtaining those commercially available(for example, Sanko Junyaku Co. Ltd., and Dainippon Seiyaku K. K.).

Porcine or human pancreatic islet that is used in the present inventioncan be isolated from porcine or human pancreas according to knownmethods (see, Staudacher C, Ricordi C, Stella M, SOcci C, Cammelli L,Ferrari G, Dicarlo V., Minerva Chir. 31: 1665-1668, 1985, Ricordi C,Finke E H, Lacy P E., Diabetes 35: 649-653, 1986, and Lakey J R T,Kobayashi N, Shapiro A M J, Ricordi C, Okitsu T: Current human isletisolation protocol. Medical Review Co., Ltd., Osaka, Japan, 2004).

As the cell cultivation method and culture conditions, commoncultivation methods and conditions can be adopted depending on the typeof cell. For example, the cultivation method includes coculture in whicha mixture of PuraMatrix of SEQ ID NO:1 and a cell is cultured in culturemedium, and preferable is culture with an insert (also referred to asculture insert) from the viewpoint of avoiding a risk of direct contactof culture medium to PuraMatrix of SEQ ID NO:1 to impart a damage toPuraMatrix of SEQ ID NO:1 itself when the culture medium is exchanged toa new one (see, FIGS. 1( a) and (b)).

An insert 2 is a cell or tissue culture equipment which has a filter 3(also referred to as membrane) at the bottom and is stacked on a culturedish 1 before seeding a cell on the insert and culturing the cell (see,FIGS. 1( a) and (b)). Examples of the material of the filter includepolyester, polycarbonate, cellulose-mixed ester and polyethyleneterephthalate, and the filter pore size is preferably 0.4 to 3.0 μmconsidering the size of cell. By using the insert, mechanical damage dueto culture medium exchange can be reduced. It can also be used formeasurement of cell secretion and drug uptake by cell. The insert iscommercially available, for example, from Becton Dickinson Biosciences,Corning Life Sciences and Millipore.

In the mixing method of a self-assembling peptide and a cell using aninsert, it is preferable in the present invention that a self-assemblingpeptide and a cell are mixed and then, culture medium is exchanged fornew one several times (e.g., 2 to 4 times). This operation exerts aneffect of converting low pH (pH 1.0 to 3.0) of a self-assembling peptideinto physiological pH (pH 6.0 to 8.0), resulting in secure gelation ofthe self-assembling peptide. Generation of physiological pH exerts aneffect of suppressing damage to cell to minimum. In culture mediumexchange, culture medium in an insert is not exchanged, and only culturemedium in a culture dish is exchanged. By this strategy, mechanicaldamage to a self-assembling peptide due to medium exchange can besuppressed to minimum.

The concentrations of a self-assembling peptide and a cell in thecultivation method of the present invention are preferably 0.5 to 1% and1×10⁵ to 3×10⁵ cells/ml, and most preferably 0.5% and 2×10⁵ cells/ml,respectively.

A medium used in the cell cultivation method of the present inventioncan be any composition providing it can grow cell, and mayadvantageously be one containing components necessary for cell culturesuch as minerals, sugars, amino acids, peptides, vitamins, organicacids, nucleic acids, pH regulators and enzymes.

Examples of the medium for hepatocyte include commercially availablemedia for hepatocyte, William's Medium E (available from SIGMA, St.Louis, Mo.), cell medium kit HCM BulletKit (product code CC-3198)(manufactured by Takara), serum free medium for hepatocyte culture:HapatoZYME-SFM (catalogue number 17705-021) (available from Invitrogen),medium for hepatocyte maintenance (Long-Term Culture Medium) (cataloguenumbers HE0306-5, HE0306) (available from KAC Co., Ltd.) and William'sMedium E containing fetal bovine serum, of which William's Medium E ispreferable in terms of cost.

To the above-described hepatocyte medium, fetal bovine serum, humanserum and various cell growth factors are preferably added when growthof cells is taken into consideration. Furthemiore, to theabove-described medium, at least one of transferrin, hydrocortisone,ascorbic acid, insulin, glutamine and nicotineamide are preferablyadded, and it is particularly preferable to add all of these types. Theaddition concentration is preferably 2.5 to 5.0 μg/ml for transferrin, 5to 10 μg/ml for hydrocortisone, 1 to 2 mmol/l for ascorbic acid, 2.5 to5 μg/ml for insulin, 2 to 5 mmol/l for glutamine and 1 to 20 mmol/l fornicotineamide, further preferably 5.0 μg/ml for transferrin, 5 μg/ml forhydrocortisone, 2 mmol/l for ascorbic acid, 5 μg/ml for insulin, 5mmol/l for glutamine and 10 mmol/l for nicotineamide.

Examples of the pancreatic islet medium include commercially availableRPMI-1640 medium (available from SIGMA), William's Medium E (availablefrom SIGMA), CMRL1066 medium (available from Invitrogen), low glucoseDMEM (available from GIPCO) and high glucose DMEM (available fromGIPCO), and preferable is low glucose DMEM from the viewpoint that sinceit can suppress insulin secretion from pancreatic islet to protect fromexhaustion of pancreatic islet cell.

To the above-described pancreatic islet medium, fetal bovine serum,human serum and various cell growth factors are preferably added whengrowth of cells is taken into consideration. Furthermore, to theabove-described medium, at least one of calcium chloride, glutamine,2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES),nicotineamide, trasylol, zinc sulfate, troglitazone and exendin-4 arepreferably added, and it is particularly preferable to add all of thesetypes. The addition concentration is preferably 1 to 3 mmol/l forcalcium chloride, 1 to 3 mmol/l for glutamine, 10 to 30 μmol/l forHEPES, 1 to 20 mmol/l for nicotineamide, 5000 to 20000 IU/l fortrasylol, 10 to 20 μmol/l for zinc sulfate, 1 to 20 μmol/l fortroglitazone and 2 to 100 nmol/l for exendin-4, further preferably 2.13mmol/l for calcium chloride, 2 mmol/l for glutamine, 20 μmol/l forHEPES, 10 mmol/l for nicotineamide, 20000 IU/l for trasylol, 16.7 μmol/lfor zinc sulfate, 10 μmol/l for troglitazone and 10 nmol/l forexendin-4.

According to the hepatocyte high-dimensional cultivation method of thepresent invention, physiologically active substances such as serumalbumin as a production component and various coagulation factorsoriginally contained in hepatocyte can be produced efficiently byculturing hepatocyte derived from porcine or human. In addition to this,it is possible to provide hepatocyte having a protein productionability, gluconeogenesis ability, urea production ability, blooddetoxification and purification ability, and amino acid, sugar and lipidmetabolic abilities that are functions of hepatocyte. According to theabove-described hepatocyte cultivation method, a detoxification abilityis maintained at a high level for a long period. For example, porcinehepatocyte cultured using an insert (on day 21 of culture) have ametabolic ability of ammonia which can be improved by about 20 to30-fold as compared with flat culture and by about 1.5 to 2.5-fold ascompared with Matrigel, have a metabolic ability of lidocaine which canbe improved by about 25 to 35-fold as compared with flat culture and by25 about 1.5 to 2.5-fold as compared with Matrigel, and have a metabolicability of diazepam which can be improved by about 20 to 30-fold ascompared with flat culture and by about 1.3 to 2.0-fold as compared withMatrigel. Human hepatocytes cultured without insert (on day 5 ofculture) have a metabolic ability of ammonia which can be improved byabout 5 to 10-fold as compared with flat culture and by about 1.1 to1.5-fold as compared with Matrigel. According to the above-describedhepatocyte cultivation method, it is possible to form spheroid(spherical and having a diameter of 100 to 120 μm), which is difficultto achieve in flat culture.

The term “spheroid” means a three-dimensional spherical cellagglomerate. Formation and/or maintenance of spheroid indicate thatphysiological functions of the above-described spheroid are similar tothose of living tissue, as compared with lone cell or irregularspheroid.

According to the hepatocyte high-dimensional cultivation method of thepresent invention, effects are obtained which are not obtained byconventional three-dimensional culture using a scaffold such asMatrigel. For example, mentioned as a morphological character isformation of a cell adhesion apparatus (also referred to asintercellular adhesion apparatus) and bile canaliculi betweenhepatocytes as features of high-dimensional culture, examined by atransmission electron microscope (see, FIGS. 2( c) and 2(d)).

The three-dimensional culture refers to a technique of sterically seed acell into a scaffold and then, culturing the cell. A conventional methodfor culturing cells having adhesiveness is characterized in that cellsare cultured on a plane of a vessel such as a petri dish(two-dimensional culture). In contrast, in the three-dimensionalcultivation method, since cells are present sterically, an environmentin organisms is simulated in a culture environment, tissue is allowed toform, and the tissue can be observed. As the three-dimensionalcultivation method, for example, collagen sandwich culture or the likeis mentioned (see, Chandra P, Lecluyse E L, Brouwer K L. Optimization ofculture conditions for determining hepatobiliary disposition oftaurocholate in sandwich-cultured rat hepatocyte. In Vitro Cell Dev BiolAnim. 2001 June; 37(6): 380-5.).

The high-dimensional culture refers to culture in which individualculture cells form “assembled” cell population while mutuallycollaborating and integrating. If a liver tissue and pancreas Langerhansislet (pancreatic islet) having high order functions can be formed byreconstruction in a growth system of cells which have been isolated andremoved from liver and pancreas, an innovative new technology leading toregeneration treatments of hepatic diseases and diabetic mellitus can beestablished. It can also be a culture technique having a possibility ofbringing an extremely large social benefit such as “industrialproduction of tissues and organs” which can be also referred to as adream of mankind, in the future.

While the three-dimensional culture means steric (three-dimensional)culture of cells which have been cultured on plane, the high-dimensionalculture is a further progressed cultivation method which is steric andin which activity as tissue is made possible. Functioning as tissue canbe explained that single or several types of cell (in liver, hepaticcell, stellate cell) constitute tissue in a culture environment, andexhibit a function of the subject organ. In such high-dimensionalculture, formation of a cell adhesion apparatus and bile canaliculi arepromoted, and functions of cells are exhibited to their maximum.

A cell adhesion apparatus 5 means an apparatus formed as a result ofrecruitment of cell skeletons around a central adhesive molecule forcell adhesion by cells, and examples thereof include adherence junction,tight junction, desmosome and hemidesmosome (see, FIGS. 2( c) and 2(d)).In multicellular organisms, individual cells are not presentindependently but mutually adhere or adhere to extracellular matrices.Mutual cell bond, and bonding of cells to extracellular matrices arecalled as cell adhesion. The former is called cell adhesion (alsoreferred to as cell-to-cell adhesion) and the latter is calledcell-matrix adhesion, and in some cases, only the former is referred toas cell adhesion in a narrow sense. The basis of cell adhesion is thatcells mutually or cells and matrices come into direct contact andachieve adhesion. However, in some cases cells have an apparatus forcell adhesion recruiting cell skeletons for adhesion. Cell adhesionoccurs by an intermolecular interaction of cell adhesion molecules, andalso an adhesion apparatus is formed around an adhesion molecule.Conditions of cell adhesion vary depending on the type of cells andtissue. In epithelium, it is common that epithelial cells are mutuallystrongly adhered, to form a specialized adhesion structure such asadherence junction, tight junction and desmosome, while, culture cellsadhered to a basement membrane as an extracellular matrix and forminghemidesmosome have a cell adhesion apparatus, thereby an adhesionstructure can be stabilized, the whole cell morphology can bemaintained, and a tissue structure can be maintained. Such an adhesionstructure also is enables to exhibit a function of preventing invasionof molecules, bacteria and viruses from outside by filling a gap betweencells. Furthermore, with such adhesion, it becomes also possible totransmit a signal into a cell and express cell characteristics such ascell differentiation and growth.

A bile canaliculi 6 is a gap formed between two hepatocytes and is atube having a diameter of about 0.5 to 1 μm (see, FIGS. 2( c) and 2(d)).Bile produced in a hepatic cell is secreted into a bile canaliculi.Formation of a bile canaliculi shows reconstruction of high-dimensionalculture, that is, tissue analogous to liver.

According to the pancreatic islet high-dimensional cultivation method ofthe present invention, insulin, a production component originallycontained in pancreatic islet, can be produced efficiently by culturingpancreatic islet derived from porcine or human. In the cultivationmethod of the present invention, the insulin production ability (highglucose culture) of the resultant pancreatic islet is preferably about 5to 15-fold as compared with flat culture. It is preferable that theinsulin production amount can be regulated by sensing the glucoseconcentration in culture medium, and it is preferable that theproduction amount is 0.4 to 0.8 μg/l (10 pancreatic islets), 0.7 to 1.5μg/l (10 pancreatic islets) and 0.5 to 1.2 μg/l (10 pancreatic islets),respectively, in culture in low glucose concentration, high glucoseconcentration and then, low glucose concentration, and it is preferablethat the difference in the insulin production amount between low glucoseconcentration and high glucose concentration being at least 0.3 μg/l (10pancreatic islets). Insulin secretion to glucose stimulation in highglucose concentration is preferably at least 1.5-fold, more preferablyat least 2.0-fold as compared with in low glucose concentration.Furthermore, it is preferable that the insulin production amount lowerswhen the glucose concentration is returned from high to low, and it ispreferable that the difference in the insulin production amount is atleast 0.3 μg/l (10 pancreatic islets). The above-described pancreaticislet cultivation method is preferable, since culture is possibletogether with successful maintenance of a shape of spheroid, that isdifficult in flat culture.

As the inspection method of cultured pancreatic islets, a method ismentioned in which the following 4 items as morphological criteria areevaluated at 5 levels (1 to 5 points) (see, literature: Matsumoto S,Qualley S A, Goel S, et al., Effect of the two-layer (University ofWisconsin solution-Perfluorochemical plus 02) method of pancreaspreservation on human islet isolation as assessed by Edmonton isolationprotocol. Transplantation 2002; 74: 1414).

1. Shape

Evaluation is performed at 5 levels of “flat: 1 point”, “approximatelyflat: 2 points”, “irregularly spherical: 3 points”, “approximatelyspherical: 4 points”, and “spherical: 5 points”.

Among them, “spherical: 5 points” is the most preferable.

2. Border Shape

Evaluation is performed at 5 levels of “irregular: 1 point”,“approximately irregular: 2 points”, “somewhat well-rounded: 3 points”,“approximately well-rounded: 4 points”, and “well-rounded: 5 points”.Among these, “well-rounded: 5 points” is most preferable.

3. Integrity

Evaluation is performed at 5 levels of “fragmented: 1 point”,“approximately fragmented: 2 points”, “somewhat solid/compact: 3points”, “approximately solid/compact: 4 points”, and “solid/compact: 5points”. Among these, “solid/compact: 5 points” is most preferable.

4. Diameter

Evaluation is performed at 5 levels of “smaller than 100 μm forindividual cultured pancreatic islets (all <100 μm): 1 point”, “100 to150 μm for individual cultured pancreatic islets: 2 points”, “125 to 175μm for individual cultured pancreatic islets: 3 points”, “150 to 200 μmfor individual cultured pancreatic islets: 4 points”, and “larger than200 μm for at least 10% of individual cultured pancreatic islets(>10%>200 μm): 5 points”. Among these, “larger than 200 μm for at least10% of individual cultured pancreatic islets: 5 points” is mostpreferable.

For pancreatic islets obtained by the cultivation method of the presentinvention using a peptide hydrogel as a scaffold, it is preferable inthe terms of morphology that the shape is at least 3 points, the bordershape is at least 3 points, the integrity is at least 3 points, thediameter is at least 3 points, and the sum of the evaluation values isat least 12 points, it is more preferable that the shape is at least 4points, the border shape is at least 4 points, the integrity is at least4 points, the diameter is at least 4 points, and the sum of theevaluation values is at least 16 points, and it is most preferable thatthe shape is at least 5 points, the border shape is at least 5 points,the integrity is at least 5 points, the diameter is at least 5 points,and the sum of the evaluation values is 20 points. Regarding thefunction of pancreatic islets obtained by the cultivation method of thepresent invention, insulin secretion in response to glucose stimulationin high glucose is preferably at least 1.5-fold, more at least 2.0-foldas compared with in low glucose.

In the above-described criteria, the shape “flat” means that when apancreatic islet is considered to be elliptic sphere, the ratio of majoraxis/minor axis is at least 10, the shape “approximately flat” meansthat the above-described ratio of major axis/minor axis is at least 5and less than 10, the shape “irregularly spherical” means that theabove-described ratio of major axis/minor axis is at least 2 and lessthan 5, the shape “approximately spherical” means that theabove-described ratio of major axis/minor axis is at least 1.2 and lessthan 2, and the shape “spherical” means that the above-described ratioof major axis/minor axis is less than 1.2.

The “irregular” border shape means that at least 90% of the border ofpancreatic islet are rough and lacking smoothness, the “approximatelyirregular” border shape means that at least 50% and less than 90% of theborder of pancreatic islet are irregular, the “somewhat well-rounded”border shape means that at least 20% and less than 50% of the border ofpancreatic islet are irregular, the “approximately well-rounded” bordershape means that at least 10% and less than 20% of the border ofpancreatic islet are irregular, and the “well-rounded” border shapemeans that less than 10% of the border of pancreatic islet areirregular.

The “fragmented” integrity means that at least 80% of all pancreaticislets have constriction, the “approximately fragmented” integrity meansthat 60 to 80% of all pancreatic islets have constriction, the “somewhatsolid/compact” integrity means that 40 to 60% of all pancreatic isletshave constriction, the “approximately solid/compact” integrity meansthat 20 to 40% of all pancreatic islets have constriction, and the“solid/compact” integrity means that at most 20% of all pancreaticislets have constriction.

The diameter “smaller than 100 μm for individual cultured pancreaticislets” means that diameters of individual cultured pancreatic isletsare all smaller than 100 μm, the diameter “100 to 150 μm for individualcultured pancreatic islets” means that diameters of individual culturedpancreatic islets are in a range of 100 to 150 μm, the diameter “125 to175 μm for individual cultured pancreatic islets” means that diametersof individual cultured pancreatic islets are in a range of 125 to 175μm, the diameter “150 to 200 μm for individual cultured pancreaticislets” means that diameters of individual cultured pancreatic isletsare in a range of 150 to 200 μm, and the diameter “larger than 200 μmfor at least 10% of individual cultured pancreatic islets” means that atleast 10% of individual cultured pancreatic islets are larger than 200μm.

Insulin secretion in response to glucose stimulation (also referred toas glucose responsive insulin secretion) means that a pancreatic isletperceives, for example, a change from low glucose concentration to highglucose concentration (glucose stimulation) in culture medium andpromotes insulin secretion. Insulin is an only hormone that acts towardlowering of blood glucose, and is secreted from .beta. cell in pancreasLangerhans islet (pancreatic islet) in response to blood glucoseconcentration. When the pancreatic islet function is excellent, insulinis secreted appropriately in response to variation in glucoseconcentration, while when the pancreatic islet function is poor, suchresponsiveness to glucose becomes poor. In cultured pancreatic islet,the ratio of the amount of insulin secreted in low glucose concentrationto the amount of insulin secreted in high glucose concentration (theamount of insulin secreted in high glucose concentration/the amount ofinsulin secreted in low glucose concentration) (represented asstimulation index; SI) is comparatively investigated, for evaluating thepancreatic islet function (see, Berger H, Knoch K P, Meisterfeld R,Jager M, Ouwendijk J, Kersting S, Saeger H D, Solimena M., Effect ofoxygenated perfluorocarbons on isolated rat pancreatic islets inculture. Cell Transplant. 2005; 14(7): 441-8), and the SI value ispreferably at least 1.5, more preferably at least 2.0.

High glucose culture refers to a culture condition of a glucoseconcentration in culture medium of 360 g/l to 450 g/l, and low glucoseculture refers to a culture condition of a glucose concentration inculture medium of 60 g/l to 100 g/l.

The cell culture obtained as described above can be suitably applied toregenerative medicine, cell preparation, useful substance production(bioreactor), investigation and research of function of tissue, organand internal organ, screening of new drugs, animal experiment substitutemethods for evaluating influences of endocrine disrupting chemicals, andcell chips, including cell transplantation, and bioartificial organssuch as bioartificial liver and bioartificial pancreatic islet.

The term “cell culture” means a suspension prepared by suspending a cellcultured using a peptide hydrogel of the present invention as ascaffold, in a medium, isotonic solution or buffering solution, togetherwith the above-described scaffold. The medium, isotonic solution orbuffering solution is appropriately selected so as to fit theabove-described cell.

In this description, the cell preparation includes the above-describedcell cultures as they are, and cell agglomerates such as pelletscondensed by filter filtration. Furthermore, the above-described cellpreparation can also be freeze-preserved with a protective agent such asDMSO added to the preparation. The cell preparation can be subjected totreatments under conditions by which proteins of pathogenic cellsdenature while leaving the function as the cell preparation, such as aheat treatment, radiation treatment and mitomycin C treatment, for saferutilization thereof.

The administration form (transplantation method) of the above-describedcell preparation using hepatocyte or pancreatic islet include, forexample, a method in which small dissection is made at lower rightabdomen, a narrow blood vessel of mesenterium is exposed and a catheteris inserted into this under direct vision and cells are transplanted, amethod in which hepatic portal is identified by echo, a catheter isneedled and cells are transplanted, and a method in which spleen isneedled directly under abdominal echo guide and transplantation intospleen is performed (see, Nagata H, Ito M, Shirota C, Edge A, McCowan TC, Fox I J: Route of hepatocyte delivery affects hepatocyte engraftmentin the spleen. Transplantation, 76(4): 732-4, 2003). Among these, themethod of performing cell transplantation by echo is more preferablebecause of less invasion, and the method of performing transplantationinto spleen by directly needling spleen under abdominal echo guide ismost preferable. The administration amount (transplantation amount) ofthe cell preparation is preferably 1×10⁸ to 1×10¹⁰ cells/individual,further preferably 5×10⁸ to 1×10¹⁰ cells/individual, and most preferably1×10⁸ to 1×10¹⁰ cells/individual. The administration amount(transplantation amount) can be appropriately altered depending on theage, body weight and symptoms of a patient to receive the therapy.

Hepatocyte and pancreatic islet obtained by the present invention can beused as sources for bioartificial liver and bioartificial pancreastargeting hepatic failures and diabetes mellitus, respectively.

The above-described bioartificial organs using hepatocyte or pancreaticislet include, for example, hybrid type artificial organs usingbioreactors of diffusion chamber type, microcapsule type and hollowfiber type obtained by enclosing a cell cultured using a peptidehydrogel of the present invention as a scaffold, into a device made of apolymer material, together with the above-described scaffold.Bioartificial organs include three forms: one mounted outside the bodyand connected to a blood vessel, one indwelled in the body and connectedto a blood vessel, and one not connected to a blood vessel and indwelledin abdominal cavity. Hepatocyte or pancreatic islet obtained by themethod of the present invention can be used in any form of bioartificialorgans.

As the bioreactor to be used as the bioartificial organ, commerciallyavailable reactors can be used. For example, various types such as HepatAssist for bioartificial liver treatment using porcine hepatocytesdeveloped mainly by Demetriou, et al. in Cedars-Sinai Medical center(Los Angeles, Calif., US) under an assist by Circe Biomedical Inc.(Lexington, Mass. US) (see, Hui T, Rozga J, Demetriou A A. JHepatobiliary Pancreat Surg 2001; 8: 1-15.), and MELS (ModularExtracorporeal Liver System) by Gerlach, et al. in German using porcinehepatocytes are known. These reactors can be used, of course, in thepresent invention, however, since there is no foothold for adhesion ofcells, there is a tendency that cell is only filled in spaces in hollowfibers or in spaces outside hollow fibers, giving a floating condition.In general, cell show a tendency that no sufficient differentialfunction is exhibited under a floating condition and furthermore,collide with surrounding cells, and easily stimulated by stress.

Therefore, preferable in the present invention are bioreactorscontaining hollow fibers, nonwoven fabric and peptide hydrogel so that afoothold can be provided for hepatocyte.

As the hollow fiber membrane, any type of membranes can be usedproviding substance exchange is not disturbed by cell adhesion to themembrane surface. Specifically, commercially available membranesconventionally used for medical treatment, for example, polysulfonemembranes, and ethylene-vinyl acetate random copolymer-saponifiedmaterial membranes (for example, trade name: Evapol, available fromKuraray Medical Inc.) and the like are preferable. Commerciallyavailable hollow fiber membranes include dialysis membranes (pore size:to 5 nm), plasma component separation membranes (pore size: 20 to 30 nm)and plasma separation membranes (pore size: 30 to 200 nm), depending onits application.

As the nonwoven fabric, those processed and modified so that cell canadhere to the fabric are preferable. Among these,polytetrafluoroethylene (PTFE) processed with poly(amino acid) urethane(PAU) is preferable from the viewpoint of easiness of processingthereof. Furthermore, those processed with basement membrane are alsopreferable (see, Katsumi Mochida, et al., Construction of artificialtissue using basement membrane formation technology, RegenerationMedicine. Vol. 5, No. 3, p. 57-63, 2006).

Using one embodiment of the bioreactor of the present invention as anexample, manufacturing processes thereof are shown in FIG. 13. Hollowfibers 12 are arranged on nonwoven fabric 11 equipped with lining 10(see, FIG. 13 (a)). Here, a slit 13 is made on the nonwoven fabric 11equipped with lining 10. This is wound in the form of roll (see, FIG. 13(b)). FIG. 13 (c) is an X-X line cross-section. The resultant roll isincorporated in a cylindrical vessel 15 equipped with liquid leakprevention parts 14 on both ends, obtaining a bioreactor 16. Thebioreactor 16 is equipped with a cell injection port 17 and a dischargeport 18 by which a cell sample can be collected, and the slit 13 isdisposed so as to communicate with the cell injection port 17.Furthermore, the bioreactor 16 is equipped with a liquid inlet 19 and aliquid outlet 20. The liquid inlet 19 and the liquid outlet 20communicate with the inside of the hollow fiber 12. The liquid inlet 19and the liquid outlet 20 constitute a blood inflow port and a bloodoutflow port, respectively, in a bioartificial organ.

The method of filling cell or cell culture in a bioreactor include, forexample, a method of culturing cell in a test tube and filling abioreactor with a necessary number of cells, and a method of processingthe inside of a bioreactor previously with PuraMatrix of SEQ ID NO:1 andculturing cells therein. In both cases, it is preferable to inject acell suspension through a cell injection port equipped on the reactorusing a 10 to 50 ml injection syringe, for filling the cell.

Treatment with a bioartificial organ using such a bioreactor ispreferably carried out by a device integrating functions of 1) real timemonitoring of inflow pressure and outflow pressure of an artificialorgan reactor, 2) actuation of alarm in generation of bubbles, and 3)warming (37° C.) of the reactor, for safe and scientific operation.

The bioreactor of the present invention can also be applied suitably foruseful substance production, investigation and research of function oftissue, organ and internal organ, screening of new drugs, and animalexperiment substitute methods for evaluating influences of endocrinedisrupting chemicals. The bioreactor of the present invention can beused as a bioreactor having a protein production ability,gluconeogenesis ability, urea production ability, blood detoxificationand purification ability, and metabolic abilities of amino acid,carbohydrate and lipid, as functions of hepatocyte. Regardingdetoxification, for example, ammonia, diazepam and lidocaine can bemetabolized. The metabolic rates for porcine hepatocyte are preferably20 to 100%, 15 to 100%, and 20 to 100%, and more preferably 30 to 100%,25 to 100%, and 25 to 100%, respectively. The ammonia metabolic rate ofhuman hepatocyte is preferably 15 to 100%, more preferably 20 to 100%.

The bioartificial pancreatic islet of the present invention can be used,for example, for insulin production which is a function of pancreaticislet. It is preferable that the insulin production amount can beregulated by sensing the glucose concentration in culture medium, andthe production amount is preferably 0.4 to 0.8 μg/l (10 pancreaticislets), 0.7 to 1.5 μg/l (10 pancreatic islets) and 0.5 to 1.2 μg/l (10pancreatic islets), respectively, in culture in low glucoseconcentration, high glucose concentration and then, low glucoseconcentration, and it is preferable that the difference in the insulinproduction amount between low glucose concentration and high glucoseconcentration is at least 0.3 μg/l (10 pancreatic islets). Insulinsecretion in response to glucose stimulation in high glucoseconcentration is preferably at least 1.5-fold, more preferably at least2.0-fold compared with in low glucose concentration. Furthermore, it ispreferable that the insulin production amount lowers when the glucoseconcentration is returned from high to low, and it is preferable thatthe difference in the insulin production amount is at least 0.3 μg/l (10pancreatic islets).

The bioartificial liver of the present invention can be used, forexample, for production of physiologically active substances such asserum albumin as a production component and various coagulation factorsoriginally contained in hepatocyte. Production of serum albumin can becarried out by purifying the resultant culture medium by methods usuallyused for purification of protein such as affinity column.

The bioartificial pancreatic islet of the present invention can be used,for example, for production of insulin, a production componentoriginally contained in pancreatic islet. Production of insulin can becarried out by purifying the resultant culture medium by methods usuallyused for purification of protein such as affinity column.

The bioartificial liver of the present invention can be used for animalexperiment substitute methods in clinical experiments such as screeningof new drugs. For example, one-third of causes for drop out of novelmedicinal nominated compounds in clinical tests is that pharmacokineticspredicted in animals using rat and dog is not reproduced in human. Thecause thereof is largely correlated with a difference in metabolismbetween species. Then, PhoenixBio Co., Ltd. started, in collaborationwith Daiichi Pharmaceutical Co., Ltd., a business for the purpose ofshortening study period and enhancing success probability of developmentof medical products, by predicting drug metabolism in human in ADME test(test of administering R1 labeled compound to animals and checkingabsorption, distribution, metabolism and excretion) using chimera micehaving human hepatocyte. As is understood from their planning of salesof 130 million yen, bioartificial livers providing prediction of data inhuman are very useful in development of medical products. However, inthis human hepatocyte chimera mouse, 80 to 90% of the liver issubstituted by human hepatocyte and the metabolic activity of rodents is10-fold of human activity and thus, even if substituted by 90%, theactivity level of the mouse is about the same as human, namely, there isa demerit that it cannot be used for evaluation of a compound showing nodifference in metabolism between species. Furthermore, since animmunodeficient SCID mouse is used as the human hepatocyte chimeramouse, there is also a problem of many cases of deaths of chimera miceeven if the administration amount is lower than the toxic dosage forusual mice. Additionally, because of 1) necessity of transplantation ofhuman hepatocytes to animals and 2) complicated procedure of breedinganimals, the project suggested by us is preferable owing to superiorityto chimera mice. It is also preferable from the viewpoint of animalprotection.

The bioartificial pancreatic islet of the present invention can betransplanted, for example, to diabetic patients whose population isincreasing every year not only in Japan but also internationally. It isnot an exaggeration to say that prevention and conquering of humandiabetic mellitus are great objectives of human beings in the 21stcentury. Therefore, many drug manufacturers are working on thedevelopment of diabetes drugs.

Recently, manufacturing of transgenic (Tg) animals has become easy, andTg animals with diabetes having various unique properties have beengenerated. For these Tg animals, establishment, maintenance,proliferation and supply of strains are laborious works. Sinceestablishment of model animals needs long period and enormous energy andtolerance, it cannot be worked on deliberately under current quickpassage of time. Furthermore, proliferation and maintenance of strainsneed manpower and equipments, and in the case of model animals whoserole has been completed, there is also a risk of loss. Because of adifference between human diabetes and diabetic model animals, there is aproblem of many cases of drop out at the clinical test stage ofdeveloped medical products. The bioartificial pancreatic islet of thepresent invention is preferable since it can solve such issues.

EXAMPLES

The present invention will be explained by examples shown below, but thepresent invention is not limited to these examples.

Example 1 Isolation and Culture of Porcine Hepatocytes

Large white male pigs (body weight: 15 to 20 kg) were used. 1.5 ml ofketalar for intramuscular injection was injected to cause sedation andthen, auricular vein was acquired and 5 mg/kg of isozol and 1 mg/kg ofmusculax were administered intravenously to obtain muscle relaxation.After endotracheal intubation, peritoneotomy was performed under generalanesthesia with sevoflurene under controlled ventilation by anartificial respirator. A liver lateral region of the pig was surgicallyexcised (about 80 g), perfusion was performed through excisedcross-section portal vein and hepatic vein. First, the excised liver wasperfused with a primary perfusion solution (sodium chloride 9 g/L,potassium chloride 0.42 g/L, sodium hydrogen carbonate 2.1 g/L, glucose0.9 g/L, HEPES 4.78 g/L, ethylenediamine tetraacetic acid (EDTA) 0.37g/L), subsequently, perfused with a secondary perfusion solutioncontaining no EDTA (sodium chloride 9 g/L, potassium chloride 0.42 g/L,sodium hydrogen carbonate 2.1 g/L, glucose 0.9 g/L, HEPES 4.78 g/L).Thereafter, perfusion of the liver, kept at 39° C., was done with adispase solution (sodium chloride 9 g/L, potassium chloride 0.42 g/L,sodium hydrogen carbonate 2.1 g/L, glucose 0.9 g/L, HEPES 4.78 g/L,dispase 8.4 g/L) (available from Godo Shusei K. K., Tokyo, Japan) andfinally, with a collagenase solution (sodium chloride 9 g/L, potassiumchloride 0.42 g/L, sodium hydrogen carbonate 2.1 g/L, glucose 0.9 g/L,HEPES 4.78 g/L, collagenase 0.5 g/L (available from Nitta Gelatin Inc.,Osaka, Japan), calcium chloride monohydrate 0.55 g/L). After perfusion,a hepatic capsule was morcellated and hepatocytes were dispersed in thecollagenase solution, and a cell suspension liquid was filtrated with 75μm mesh, and this was centrifuged at a low speed (50 g, 2 minutes). Thepellet was re-suspended in a primary washing liquid (sodium chloride 7g/L, potassium chloride 0.46 g/L, calcium chloride monohydrate 0.13 g/L,HEPES 2.38 g/L, bovine serum albumin (BSA) 1.0 g/L (available fromSIGMA), magnesium chloride hexahydrate 0.1 g/L, magnesium chlorideheptahydrate 0.1 g/L, deoxyribonuclease (DNaseI) 0.1 g/L (available fromRoche Mannheim Germany)), and furthermore, centrifuged at low speed (50g, 75 seconds). This operation was repeated three times and then, thepellet was suspended in a secondary washing liquid (sodium chloride 7g/L, potassium chloride 0.46 g/L, calcium chloride monohydrate 0.13 g/L,HEPES 2.38 g/L, bovine serum albumin 1.0 g/L (available from SIGMA),magnesium chloride hexahydrate 0.1 g/L, magnesium chloride heptahydrate0.1 g/L). The isolated porcine hepatocytes were suspended in ahepatocyte culture medium (William's Medium E (available from SIGMA),10% fetal bovine serum (available from SIGMA), insulin 1×10-⁷ mol/L(GIBCOBRL13007-018) (available from GIBCO BRL), epithelial cell growthfactor (EGF) 25 μg/L (available from SIGMA), dexametazone 1×10-⁶ mol/L(available from SIGMA), penicillin 1×10-⁵ U/L and streptomycin 1×10-⁵μg/L) and then, cultured with or without insert.

(Culture without Insert)

Cells were seeded (2×10⁵ cells/well) on 12-well plates coated withPuraMatrix of SEQ ID NO:1 (group A) and 12-well plates coated withcollagen type I (monolayer) (group B) or Matrigel (group C) (BIOCOAT,available from Becton Dickinson Labware) and then, cultured for 18 hoursat 37° C. under 5% CO₂ to obtain porcine hepatocytes which were used inthe following Examples 2 to 6. Exchange of the medium was carried outevery two days.

(Culture Using Insert)

An insert (available from Becton Dickinson BioScience) was used, andcell culture was carried out on a filter of the insert. In this method,the insert can be removed in exchanging the culture medium for newmedium. As a result, mechanical damage on a scaffold due to mediumexchange can be alleviated. PuraMatrix of SEQ ID NO:1 (group A),collagen type I (monolayer) (collagen is added on an insert, permeatedin culture medium at room temperature overnight and then, cells areseeded) (group B), Matrigel (Matrigel is added on an insert, permeatedin culture medium at room temperature overnight and then, cells areseeded) (group C), or collagen sandwich (hepatocytes are cultured,intervening between collagen monolayers: first, a collagen monolayer isplaced and then, hepatocytes are seeded thereon, finally, a collagenmonolayer is placed thereon, providing an environment similar tothree-dimensional culture to cells) (group D), and cells were seeded(2×10⁵ cells/insert) and then, cultured for 18 hours at 37° C. under 5%CO₂ to obtain porcine hepatocytes which were used in the followingExamples 2 to 6. Exchange of the medium was carried out every two days.In culture using PuraMatrix of SEQ ID NO:1 as a scaffold (group A), amethod of sowing cells by the following processes was adopted.

1) PuraMatrix of SEQ ID NO:1 was diluted to 0.5% with phosphate bufferedsaline (PBS) at room temperature (RT).

2) Hepatocytes were suspended (2×10⁵ cells/ml) in PBS (RT), and thenmixed with equal volume of the above-described PuraMatrix of SEQ ID NO:1of 0.5% concentration in a 200 μl tube, where pipetting was performed inshort while (within 5 seconds). Then, a mixture of hepatocytes suspendedwith PuraMatrix of SEQ ID NO:1+PBS was seeded at the bottom of theinsert.

At this step, a hepatocyte culture medium was filled in advance, in aculture dish 12 well-plate into which an insert is to be placed, inorder to avoid a damage of cells due to low pH of PuraMatrix of SEQ IDNO:1, as much as possible. Thereby, pH is adjusted to physiological pH.

3) An insert was placed for 5 minutes at RT and then, the insert wasremoved, and the hepatocyte culture medium in the culture dish 12-wellplate was exchanged for new one quickly. Then, the inset was placedthereon.

4) PuraMatrix of SEQ ID NO:1 was left as it was at RT for 10 minutes toadjust its pH.

5) Furtheimore, the insert was removed at RT, and the hepatocyte culturemedium in the culture dish 12-well plate was 10 exchanged for new onequickly. Then, the inset was placed thereon.

6) Operations 4) to (5) were carried out again and then, culture wasperformed in a culture vessel at 37° C. under 5% CO2 without any change.

Example 2 Morphological Study of Cultured Porcine Hepatocyte byPhase-Contrast Microscope and Electron Microscope

(Culture without Insert)

Culture conditions of cultured porcine hepatocytes were observed on days1, 7 and 14 of culture with a-phase-contrast microscope and comparedbetween groups A and B. In the group using PuraMatrix of SEQ ID NO:1(group A), cells kept a spherical morphology, and individual cellsgradually formed an agglomerate (see, Photographs 1, 2 and 3 in FIG. 2(a)). Culture conditions of porcine hepatocytes on day 5 of culture wereobserved with an electron microscope, as a result, formation ofthree-dimensional spheroid by hepatocytes was confirmed (FIG. 2 (b),Photograph 1: only PuraMatrix of SEQ ID NO:1, Photograph 2: porcinehepatocytes covering over PuraMatrix of SEQ ID NO:1, Photograph 3:porcine hepatocytes forming three-dimensional spheroid on PuraMatrix ofSEQ ID NO:1, Photograph 4: porcine hepatocytes initiating to formthree-dimensional spheroid on PuraMatrix of SEQ ID NO:1, Photograph 5:lower magnification image of Photograph 4, Photograph 6: porcinehepatocytes forming three-dimensional spheroid on PuraMatrix of SEQ IDNO:1). Recently, it has been known that when three-dimensional cultureof cells is performed on scaffold, cell functions are improved andmaintained. Since cultured porcine hepatocytes formed three-dimensionalspheroid, it is strongly indicated that culturing porcine hepatocyteswith PuraMatrix of SEQ ID NO:1 is useful for improvement of functions.In contrast, porcine hepatocytes cultured with collagen type I(monolayer) (group B) adhered in flat form to the surface of the cultureflask (see, Photograph 4 in FIG. 2 (a)). The cell number decreased withtime and formation of spheroid was not observed (see, Photographs 4, 5and 6 in FIG. 2 (a)). As a result, it was clarified that it is moresuitable for culture of porcine hepatocyte to use PuraMatrix of SEQ IDNO:1.

(Culture with Insert)

Culture conditions of cultured porcine hepatocytes were observed on day3 of culture with a transmission electron microscope (see, FIGS. 2( c)and (d)). In the group using PuraMatrix of SEQ ID NO:1 (group A), a celladhesion apparatus 5 and a bile canaliculi 6 were formed betweenhepatocytes, with a transmission electron microscope. This shows thatthe cell cultivation is done higher-dimensionally. In contrast, sinceformation of them was not seen in porcine hepatocytes cultured in thegroups B, C and D. As a result, it was clarified that it is moresuitable for culture of porcine hepatocyte to use PuraMatrix of SEQ IDNO:1 and an insert.

Example 3 Measurement of Viability of Porcine Hepatocyte

(Culture without Insert)

In a survival test (live/dead test) (kit name: Live/Dead Double StainingKit, catalogue No. QIA76, available from Cosmo Bio Co., Ltd. (Koto ward,Tokyo), the viability of porcine hepatocytes on days 1, 7 and 14 ofculture was measured and compared for groups A and B. The live cells areentirely stained green, and the dead cells are entirely stained red. Inthe group using PuraMatrix of SEQ ID NO:1 (group A), most cellsexhibited a green color, namely, survival of them was evident (see,Photographs 1, 2 and 3 in FIG. 3). In contrast, porcine hepatocytescultured with collagen type I (monolayer) (group B) exhibited a greencolor on day 1 of culture, thereby indicating survival (see, Photograph4 in FIG. 3). However, on days 7 and 14 of culture, only cellsexhibiting a red color, indicating dead cell, were present (see,Photographs 5 and 6 in FIG. 3). Therefore, it was clarified that it ismore suitable for culture of porcine hepatocyte to use PuraMatrix of SEQID NO:1 Since the appended Photographs are converted into white andblack two tones, dyed colors are not expressed.

Example 4 Measurement of Metabolic Ability of Ammonia

(Culture without Insert)

The metabolic abilities of ammonia in cultured porcine hepatocyte weremeasured and compared between groups A and B. Ammonium sulfate (0.56 mM)was added to porcine hepatocyte media on days 1, 7 and 14 of culture,and the concentration of ammonia in the media after 24 hours wasmeasured (FUJI DRI-CHEM SLIDE (Fuji Co., Tokyo, Japan)), and themetabolic rate was calculated. The results are shown in FIG. 4 (a) andTable 1(a). In the group using PuraMatrix of SEQ ID NO:1 (group A), theammonia metabolic rate was significantly better as compared withcollagen type I (monolayer) (group B). The expression “culture mediumonly” in the graph and table means that culture medium only, containingno cells, is measured, and the significant difference was tested, usingANNOVA.

TABLE 1(a) Culture Standard Standard medium Standard Group A deviationGroup B deviation only deviation Day 1 of culture 58.6 2.0 53.1 2.0 0.00.0 Day 7 of culture 43.8 2.2 16.6 1.5 0.0 0.0 Day 14 of 26.4 3.0 9.01.3 0.0 0.0 culture(Culture without Insert)

The metabolic abilities of ammonia in cultured porcine hepatocyte weremeasured and compared between groups A, B and C.

Ammonium sulfate (0.56 mM) was added to porcine hepatocyte media on days1, 7 and 14 of culture, and the concentration of ammonia in the mediaafter 24 hours was measured (FUJI DRI-CHEM SLIDE), and the metabolicrate was calculated. The results are shown in FIG. 4( b) and Table 1(b).In the group using PuraMatrix of SEQ ID NO:1 (group A), the ammoniametabolic rate was significantly better as compared with collagen type I(monolayer) (group B) and Matrigel (group C). The expression “culturemedium only” in the graph and table means that culture medium only,containing no cells, is measured, and the significant difference wastested, using ANNOVA.

TABLE 1b Culture Standard Standard Standard medium Standard Group Adeviation Group C deviation Group B deviation only deviation Day 1 of59.8 1.9 57.5 2.5 56.7 2.7 0.0 0.0 culture Day 7 of 44.3 2.4 39.9 2.917.2 1.8 0.0 0.0 culture Day 14 of 33.3 2.4 22.7 2.7 8.7 2.4 0.0 0.0culture(Culture with Insert)

The metabolic abilities of ammonia in cultured porcine hepatocyte weremeasured and compared between groups A, B, C and D. Ammonium sulfate(0.56 mM) was added to porcine hepatocyte media on days 1, 7, 14 and 21of culture, and the concentration of ammonia in the media after 24 hourswas measured (FUJI DRI-CHEM SLIDE), and the metabolic rate wascalculated. The results are shown in FIG. 4( c) and Table 1(c). In thegroup using PuraMatrix of SEQ ID NO:1 (group A), the ammonia metabolicrate was significantly better as compared with collagen type I(monolayer) (group B), Matrigel (group C) and collagen sandwich (groupD). The expression “culture medium only” in the graph and table meansthat culture medium only, containing no cells, is measured, and thesignificant difference was tested, using ANNOVA.

TABLE 1(c) Culture medium Group A Group C Group D Group B only Day 1 of61.6 60.2 59.3 59.0 0.0 culture Day 7 of 49.6 44.6 41.7 18.6 0.0 cultureDay 14 of 38.2 33.8 32.3 7.4 0.0 culture Day 21 of 36.1 25.5 23.4 1.60.0 culture Standard Standard Standard Standard Standard deviationdeviation deviation deviation deviation Day 1 of 1.9 1.6 2.1 1.9 0.0culture Day 7 of 1.6 2.9 1.4 1.8 0.0 culture Day 14 of 1.9 2.4 2.0 1.60.0 culture Day 21 of 2.9 2.5 1.4 0.9 0.0 culture

Example 5 Measurement of Metabolic Ability of Lidocaine

(Culture without Insert)

The metabolic abilities of lidocaine in cultured porcine hepatocyte weremeasured and compared between groups A and B. Lidocaine (1 mg/ml) wasadded to porcine hepatocyte media on days 1, 7 and 14 of culture, andthe concentration of lidocaine in the media after 10 24 hours wasmeasured (entrusted to SRL K. K.), and the metabolic rate wascalculated. The results are shown in FIG. 5 and Table 2. In the groupusing PuraMatrix of SEQ ID NO:1 (group A), the lidocaine metabolic ratewas significantly better as compared with collagen type I (monolayer)(group B). The expression “culture medium only” in the graph and tablemeans that culture medium only, containing no cells, is measured, andthe significant difference was tested, using ANNOVA.

TABLE 2(a) Culture Standard Standard medium Standard Group A deviationGroup B deviation only deviation Day 1 of culture 40.0 1.8 38.8 3.4 0.00.0 Day 7 of culture 31.9 4.0 11.6 1.0 0.0 0.0 Day 14 of 19.1 3.0 8.11.7 0.0 0.0 culture(Culture without Insert)

The metabolic abilities of lidocaine in cultured porcine hepatocyte weremeasured and compared between groups A, B and C. Lidocaine (1 mg/ml) wasadded to porcine hepatocyte media on days 1, 7 and 14 of culture, andthe concentration of lidocaine in the media after 24 hours was measured(entrusted to SRL K. K.), and the metabolic rate was calculated. Theresults are shown in FIG. 5( b) and Table 2(b). In the group usingPuraMatrix of SEQ ID NO:1 (group A), the lidocaine metabolic rate wassignificantly better as compared with collagen type I (monolayer) (groupB) and Matrigel (group C). The expression “culture medium only” in thegraph and table means that culture medium only, containing no cells, ismeasured, and the significant difference was tested, using ANNOVA.

TABLE 2b Culture Standard Standard Standard medium Standard Group Adeviation Group C deviation Group B deviation only deviation Day 1 of40.8 2.1 39.1 2.2 38.4 2.4 0.0 0.0 culture Day 7 of 32.2 3.3 27.9 2.69.7 2.9 0.0 0.0 culture Day 14 of 21.0 1.5 15.5 2.5 6.1 2.8 0.0 0.0culture(Culture with Insert)

The metabolic abilities of lidocaine in cultured porcine hepatocyte weremeasured and compared between groups A, B, C and D. Lidocaine (1 mg/ml)was added to porcine hepatocyte media on days 1, 7, 14 and 21 ofculture, and the concentration of lidocaine in the media after 24 hourswas measured (entrusted to SRL K. K.), and the metabolic rate wascalculated. The results are shown in FIG. 5( b) and Table 2(b). In thegroup using PuraMatrix of SEQ ID NO:1 (group A), the lidocaine metabolicrate was significantly better as compared with collagen type I(monolayer) (group B), Matrigel (group C) and collagen sandwich (groupD). The expression “culture medium only” in the graph and table meansthat culture medium only, containing no cells, is measured, and thesignificant difference was tested, using ANNOVA.

TABLE 2(c) Culture medium Group A Group C Group D Group B only Day 1 of44.0 43.2 42.6 42.1 0.0 culture Day 7 of 36.9 31.7 28.1 10.2 0.0 cultureDay 14 of 32.4 25.3 22.3 5.0 0.0 culture Day 21 of 30.2 20.5 18.5 0.90.0 culture Standard Standard Standard Standard Standard deviationdeviation deviation deviation deviation Day 1 of 2.0 1.6 2.1 1.6 0.0culture Day 7 of 1.6 1.8 2.5 2.0 0.0 culture Day 14 of 1.8 3.0 1.4 1.60.0 culture Day 21 of 1.3 1.4 1.3 0.3 0.0 culture

Example 6 Measurement of Metabolic Ability of Diazepam

(Culture without Insert)

The metabolic abilities of diazepam in cultured porcine hepatocyte weremeasured and compared between groups A and B. Diazepam (1 pg/ml) wasadded to porcine hepatocyte media on days 1, 7 and 14 of culture, andthe concentration of diazepam in the media after 24 hours was measured(entrusted to SRL K. K.), and the metabolic rate was calculated. Theresults are shown in FIG. 6( a) and Table 3(a). In the group usingPuraMatrix of SEQ ID NO:1 (group A), the diazepam metabolic rate wassignificantly better as compared with collagen type I (monolayer) (groupB). The expression “culture medium only” in the graph and table meansthat culture medium only, containing no cells, is measured, and thesignificant difference was tested, using ANNOVA.

TABLE 3(a) Culture Standard Standard medium Standard Group A deviationGroup B deviation only deviation Day 1 of culture 39.9 2.8 37.2 2.0 0.00.0 Day 7 of culture 34.6 4.0 13.9 2.0 0.0 0.0 Day 14 of 24.0 3.6 5.81.7 0.0 0.0 culture(Culture without Insert)

The metabolic abilities of diazepam in cultured porcine hepatocyte weremeasured and compared between groups A, B and C. Diazepam (1 pg/ml) wasadded to porcine hepatocyte media on days 1, 7 and 14 of culture, andthe concentration of diazepam in the media after 24 hours was measured(entrusted to SRL K. K.), and the metabolic rate was calculated. Theresults are shown in FIG. 6( b) and Table 3(b). In the group usingPuraMatrix of SEQ ID NO:1 (group A), the diazepam metabolic rate wassignificantly better as compared with collagen type I (monolayer) (groupB) and Matrigel (group C). The expression “culture medium only” in thegraph and table means that culture medium only, containing no cells, ismeasured, and the significant difference was tested, using ANNOVA.

TABLE 3b Culture Standard Standard Standard medium Standard Group Adeviation Group C deviation Group B deviation only deviation Day 1 of39.0 1.9 38.8 3.1 36.8 2.5 0.0 0.0 culture Day 7 of 35.3 2.6 31.1 2.711.4 2.8 0.0 0.0 culture Day 14 of 24.9 2.2 21.0 2.9 6.7 2.8 0.0 0.0culture(Culture with Insert)

The metabolic abilities of diazepam in cultured porcine hepatocyte weremeasured and compared between groups A, B, C and D. Diazepam (1 pg/ml)was added to porcine hepatocyte media on days 1, 7, 14 and 21 ofculture, and the concentration of diazepam in the media after 24 hourswas measured (entrusted to SRL K. K.), and the metabolic rate wascalculated. The results are shown in FIG. 6( c) and Table 3(c). In thegroup using PuraMatrix of SEQ ID NO:1 (group A), the diazepam metabolicrate was significantly better as compared with collagen type I(monolayer) (group B), Matrigel (group C) and collagen sandwich (groupD). The expression “culture medium only” in the graph and table meansthat culture medium only, containing no cells, is measured, and thesignificant difference was tested, using ANNOVA.

TABLE 3(c) Culture medium Group A Group C Group D Group B only Day 1 of41.1 40.2 39.5 39.2 0.0 culture Day 7 of 37.3 33.3 32.8 13.2 0.0 cultureDay 14 of 28.7 25.6 23.0 6.3 0.0 culture Day 21 of 27.1 20.7 17.7 1.10.0 culture Standard Standard Standard Standard Standard deviationdeviation deviation deviation deviation Day 1 of 1.4 2.1 1.8 1.9 0.0culture Day 7 of 2.6 2.8 2.5 2.5 0.0 culture Day 14 of 1.5 2.8 2.4 3.00.0 culture Day 21 of 2.8 2.7 3.3 0.6 0.0 culture

Example 7 Isolation and Culture of Human Hepatocyte

Donor liver judged to be unsuitable for transplantation in the UnitedStates was obtained from National Disease Research Interchange via HUMAN& ANIMAL BRIDGING RESEARCH ORGANIZATION laboratory (Ichikawa city, Chibaprefecture, responsible person: Mr. Satoshi Suzuki) in the form of liverblock (130 g) which was then separated into hepatocyte in the samemanner as for the above-described isolation of porcine hepatocyte inExample 1. The isolated hepatocytes were seeded (5×10⁵ cells/well) on6-well plates (without insert) coated with PuraMatrix of SEQ ID NO:1(group A) or collagen type I (monolayer) (group B) and then, culturedfor 18 hours at 37° C. under 5% CO₂ to obtain human hepatocytes whichwere used in the following Examples 8 and 9. Exchange of the medium wascarried out every two days.

Example 8 Morphological Study of Cultured Human Hepatocyte byPhase-Contrast Microscope and Electron Microscope

Culture conditions of cultured human hepatocytes were observed on days 1and 5 of culture with a phase-contrast microscope and compared betweengroups A and B. In the group using PuraMatrix of SEQ ID NO:1 (group A),cells kept a spherical morphology, and individual cells gradually formedan agglomerate (see, Photographs 3 and 4 in FIG. 7( a)). Cultureconditions of human hepatocytes on day 5 of culture were observed withan electron microscope, as a result, formation of three-dimensionalspheroid by hepatocytes was confirmed (FIG. 7( b), Photograph 1: humanhepatocytes covering over PuraMatrix of SEQ ID NO:1, Photograph 2: humanhepatocytes initiating to form three-dimensional spheroid on PuraMatrixof SEQ ID NO:1, Photograph 3: magnification image of Photograph 1,Photograph 4: human hepatocytes forming three-dimensional spheroid onPuraMatrix of SEQ ID NO:1). Recently, it has been known that whenthree-dimensional culture of cells is performed on scaffold, cellfunctions are improved and maintained. Since cultured human hepatocytesformed three-dimensional spheroid, it is strongly indicated thatculturing human hepatocytes with PuraMatrix of SEQ ID NO:1 is useful forimprovement of functions. In contrast, human hepatocytes cultured withcollagen type I (monolayer) (group B) adhered in flat form to thesurface of the culture flask (see, Photograph 1 in FIG. 7( a)). The cellnumber decreased with time and formation of spheroid was not observed(see, Photographs 1 and 2 in FIG. 7( a)). As a result, it was clarifiedthat it is more suitable for culture of human hepatocyte to usePuraMatrix of SEQ ID NO:1.

Example 9 Measurement of Metabolic Ability of Ammonia

The metabolic abilities of ammonia in cultured human hepatocytes weremeasured and compared between groups A and B. Ammonium sulfate (0.56 mM)was added to human hepatocyte media on days 1, 3 and 5 of culturewithout insert, and the concentration of ammonia in the media after 24hours was measured (FUJI DRI-CHEM SLIDE), and the metabolic rate wascalculated. The results are shown in FIG. 8( a) and Table 4(a). In thegroup using PuraMatrix of SEQ ID NO:1 (group A), the ammonia metabolicrate was significantly better as compared with collagen type I(monolayer) (group B). The expression “culture medium only” in the graphand table means that culture medium only, containing no cells, ismeasured, and the significant difference was tested, using ANNOVA.

TABLE 4(a) Culture Standard Standard medium Standard Group A deviationGroup B deviation only deviation Day 1 of culture 45.0 2.9 39.0 3.2 0.00.0 Day 3 of culture 32.8 2.8 19.7 2.6 0.0 0.0 Day 5 of 20.4 3.4 3.6 1.90.0 0.0 culture

The metabolic abilities of ammonia in cultured human hepatocyte weremeasured and compared between groups A, B and C. Ammonium sulfate (0.56mM) was added to human hepatocyte media on days 1, 3 and 5 of culturewithout insert, and the concentration of ammonia in the media after 24hours was measured (FUJI DRI-CHEM SLIDE), and the metabolic rate wascalculated. The results are shown in FIG. 8( b) and Table 4(b). In thegroup using PuraMatrix of SEQ ID NO:1 (group A), the ammonia metabolicrate was significantly better as compared with collagen type I(monolayer) (group B) and Matrigel (group C). The expression “culturemedium only” in the graph and table means that culture medium only,containing no cells, is measured, and the significant difference wastested, using ANNOVA.

TABLE 4(b) Culture medium Group A Group C Group B only Day 1 of 45.044.2 39.0 0.0 culture Day 3 of 32.8 30.6 19.7 0.0 culture Day 5 of 20.418.2 3.6 0.0 culture Standard Standard Standard Standard deviationdeviation deviation deviation Day 1 of 2.9 1.9 3.2 0.0 culture Day 3 of2.8 1.6 2.6 0.0 culture Day 5 of 3.4 1.9 1.9 0.0 culture

Example 10 Culture of Porcine Pancreatic Islet

Large white male pigs (body weight: 15 to 20 kg) were used similarly toExample 1. 1.5 ml of ketalar for intramuscular injection was injected toinduce sedation and then, auricular vein was acquired and 5 mg/kg ofisozol and 1 mg/1 g of musculax were administered intravenously toachieve muscle relaxation. After endotracheal intubation, peritoneotomywas performed under general anesthesia with sevoflurene under controlledventilation by an artificial respirator. A process of isolation ofporcine pancreatic islet was carried out according to the method forisolation of human pancreatic islet (see, Lakey J R T, Kobayashi N,Shapiro A M J, Ricordi C, Okitsu T: Current human islet isolationprotocol. Medical Review Co., Ltd., Osaka, Japan, 2004), and followed amethod in a published literature (Yonekawa Y, Matsumono S, Okitsu T,Arata T, Iwanaga Y, Noguchi H, Nagata H, O'Neil J J, Tanaka K: Effectiveislet isolation method with extremely high islet yields from adult pigs.Cell Transplant. 14(10): 757-62, 2005). Those skilled in the art arecapable of isolating pancreatic islet by referring to this literature.The isolated porcine pancreatic islets were suspended in William'sMedium E supplemented with 10% fetal bovine serum, insulin 10-⁷ mol/l(available from SIGMA), dexametazone 10-⁶ mol/l (available from SIGMA),EGF 25 μg/ml, nicotineamide 10 mM (available from SIGMA), and antibioticpenicillin G/streptomycin (available from SIGMA), and 10 pancreaticislets were seeded on 6-well plates (without insert) coated withPuraMatrix of SEQ ID NO:1 (group A) and collagen type I (monolayer)(group B), respectively and then, cultured for 18 hours at 37° C. under25 5% CO2.

Example 11 Morphological Study of Cultured Porcine Pancreatic Islet byElectron Microscope

Culture conditions of cultured porcine pancreatic islets were observedon day 5 of culture with an electron microscope and compared betweengroups A and B (see, FIG. 9). Scale bars in FIG. 9 show 100 pm in boththe groups A and B. In the group using PuraMatrix of SEQ ID NO:1 (groupA), cells kept spherical morphology, and in terms of morphology: 1.shape “spherical: 5 points”, 2. border shape “well-rounded: 5 points”,3. integrity “solid/compact: 5 points” and 4. diameter “125 to 175 pmfor whole cultured pancreatic islets: 3 points”, sum thereof being 18points (see, group A in FIG. 9). In contrast, porcine pancreatic isletscultured with collagen type I (monolayer) (group B) had irregular shape,and in terms of morphology: 1. shape “irregularly spherical: 3 points”,2. Border shape “approximately irregular: 2 points”, 3. integrity“approximately fragmented: 2 points” and 4. diameter “less than 100 pmfor whole cultured pancreatic islets: 1 point”, sum thereof being 8points (see, group B in FIG. 9).

Example 12 Measurement of Insulin Production Ability

Insulin production abilities of cultured porcine pancreatic islet weredetermined and compared between groups A and B. Using low glucose DMEM(glucose concentration: 100 g/l) (available from 25 GIBCO, Oakland,N.J.) supplemented with 10% FCS, nicotineamide 10 mM and penicillinG/streptomycin, cells were cultured for 24 hours until 60% confluency(low glucose). Then, the medium was changed for high glucose DMEM(glucose concentration: 450 g/l) (available from GIBCO, Oakland, N.J.)supplemented with 10% FCS, nicotineamide 10 mM and penicillinG/streptomycin, and cells were cultured for 6 hours (high glucose).After high glucose culture, the medium was exchanged for low glucoseDMEM and cells were cultured for 6 hours (low glucose (after)). Aftereach culture operation, the production amount of insulin in the culturemedium (μg/l) was determined by an immunostaining method using a rabbitanti-human insulin antibody (available from DakoCytomation K. K., Kyoto,Japan). The results of staining are shown in FIG. 10 and Table 5. On day5 of culture, in the group using PuraMatrix of SEQ ID NO:1 (group A),the amount of insulin secreted increased in high glucose concentration,and the amount of insulin secreted decreased in low glucoseconcentration. From these results, it was clarified that pancreaticislet obtained by the present invention retain a function of secretinginsulin by response to the glucose concentration for a longer period oftime, as compared with pancreatic islet cultured using collagen type I(monolayer) (group B) according to a conventional method. The expression“culture medium only” in the graph and table means that culture mediumonly, containing no cells, is measured, and the significant differencewas tested, using ANNOVA.

TABLE 5 Group A Group B Day 0 of culture Low glucose 0.80 0.80 Highglucose 1.62 1.61 Low glucose (after) 1.01 0.99 Culture medium only 0.000.00 Day 3 of culture Low glucose 0.80 0.50 High glucose 1.48 0.76 Lowglucose (after) 0.90 0.65 Culture medium only 0.00 0.00 Day 5 of cultureLow glucose 0.69 0.37 High glucose 1.24 0.40 Low glucose (after) 0.780.40 Culture medium only 0.00 0.00 Day 7 of culture Low glucose 0.490.10 High glucose 0.90 0.10 Low glucose (after) 0.71 0.10 Culture mediumonly 0.00 0.00 unit: (μg/l)

Example 13 Culture of Human Pancreatic Islet

Healthy isolated human pancreatic islets provided from AlbertaUniversity in Canada (those skilled in the art can obtain them fromCanada, Alberta University, human pancreatic islet transplantationprogram, Dr. Jonathan R T. Lakey) were seeded in a T25 10 culture flask.Human pancreatic islets were suspended in low glucose DMEM supplementedwith 10% fetal bovine serum, insulin 10-⁷ mol/l, dexametazone 10-⁶mol/l, EGF 25 μg/ml, nicotineamide 10 mM and antibiotic penicillinG/streptomycin, and 10 pancreatic islets were seeded on 6-well plates(without insert) coated with PuraMatrix of SEQ ID NO:1 (group A) orcollagen type I (monolayer) (group B) and then, cultured for 18 hours at37° C. under 5% CO2.

Example 14 Morphological Study of Cultured Human Pancreatic Islet byElectron 10 Microscope

Culture conditions of cultured human pancreatic islets were observed onday 5 of culture with an electron microscope and compared between groupsA and B (see, FIG. 11). Scale bars in FIG. 11 show 50 pm (group A) and100 pm (group B), respectively. In the group using PuraMatrix of SEQ IDNO:1 (group A), cells kept spherical morphology, and in terms ofmorphology: 1. shape “spherical: 5 points”, 2. border shape “irregularlyspherical: 3 points”, 3. integrity “solid/compact: 5 points” and 4.diameter “125 to 175 pm for whole cultured pancreatic islets: 3 points”,sum thereof being 16 points (see, group A in FIG. 11). In contrast,porcine pancreatic islets cultured with collagen type I (monolayer)(group B) had irregular shape, and in terms of morphology: 1. shape“approximately flat: 2 points”, 2. border shape “irregular: 1 point”, 3.integrity “somewhat solid/compact: 3 points” and 4. diameter “125 to 175pm for whole cultured pancreatic islets: 3 point”, sum thereof being 9points (see, group B in FIG. 11).

Example 15 Measurement of Insulin Production Ability

Insulin production abilities of cultured human pancreatic islet weredetermined and compared between groups A and B. Using low glucose DMEM(glucose concentration: 100 g/l) (available from GIBCO, Oakland, N.J.)supplemented with 10% FCS, nicotineamide 10 mM and penicillinG/streptomycin, cells were cultured for 24 hours until 60% confluency(low glucose). Then, the medium was changed for high glucose DMEM(glucose concentration: 450 g/l) (available from GIBCO, Oakland, N.J.)supplemented with 10% FCS, nicotineamide 10 mM and penicillinG/streptomycin, and cells were cultured for 6 hours (high glucose).After high glucose culture, the medium was exchanged for low glucoseDMEM and cells were cultured for 6 hours (low glucose (after)). Afterrespective culture operations, the production amount of insulin in theculture medium (μg/l) was determined by an immunostaining method using arabbit anti-human insulin antibody (available from DakoCytomation K. K.,Kyoto, Japan). The results of staining are shown in FIG. 12 and Table 6.On day 7 of culture, in the group using PuraMatrix of SEQ ID NO:1 (groupA), the amount of insulin secreted increased in high glucoseconcentration, and the amount of insulin secreted decreased in lowglucose concentration. From these results, it was clarified thatpancreatic islet obtained by the present invention retain a function ofsecreting insulin by response to the glucose concentration for a longerperiod of time, as compared with pancreatic islet cultured using collagen type I (monolayer) (group B) according to a conventional method.The expression “culture medium only” in the graph and table means thatculture medium only, containing no cells, is measured, and thesignificant difference was tested, using ANNOVA.

TABLE 6 Group A Group B Day 1 of culture Low glucose 0.60 0.57 Highglucose 1.89 1.73 Low glucose (after) 1.13 1.29 Culture medium only 0.000.00 Day 3 of culture Low glucose 0.58 0.78 High glucose 1.68 1.32 Lowglucose (after) 1.11 1.19 Culture medium only 0.00 0.00 Day 5 of cultureLow glucose 0.51 0.57 High glucose 1.43 0.66 Low glucose (after) 1.100.79 Culture medium only 0.00 0.00 Day 7 of culture Low glucose 0.410.18 High glucose 0.76 0.12 Low glucose (after) 0.54 0.10 Culture mediumonly 0.00 0.00 unit: (μg/l)

INDUSTRIAL APPLICABILITY

According to the present invention, by culturing a cell such as porcinehepatocyte, human hepatocyte, porcine pancreatic islet or humanpancreatic islet using a peptide hydrogel as a scaffold,high-dimensional culture can be carried out for a long period underconditions where cell survival, cell morphology and cell functions aremaintained.

The invention claimed is:
 1. A method for culturing a primary cell in acell culture, comprising: providing differentiated functional primarycells selected from the group consisting of porcine pancreatic isletsand human pancreatic islets; culturing the differentiated functionalprimary cells using an insert and a self-assembling peptide hydrogel ofSEQ ID NO:1; evaluating the differentiated functional primary cells inthe cell culture based on pancreatic islet morphology criteria;assigning evaluation values of 1 to 5 points for each of the criteriaconsisting of: shape having evaluation values of 1 point for flat shape,2 points for approximately flat shape, 3 points for irregularlyspherical shape, 4 points for approximately spherical shape, and 5points for spherical shape; border integrity having evaluation values of1 point for irregular border, 2 points for approximately irregularborder, 3 points for somewhat well-rounded border, 4 points forapproximately well-rounded border and 5 points for well-rounded border;cell integrity having evaluation values of 1 point for fragmentedintegrity, 2 points for approximately fragmented integrity, 3 points forsomewhat solid/compact integrity, 4 points for approximatelysolid/compact integrity, and 5 points for solid/compact integrity; andcell diameter having evaluation values of 1 point for smaller than 100μm diameter (all <100 μm), 2 points for 100 to 150 μm diameter, 3 pointsfor 125 to 175 μM diameter, 4 points for 150 to 200 μm diameter, and 5points for larger than 200 μm for at least 10% of individual culturedpancreatic islets (>10%>200 μm); and totaling the evaluation valuepoints resulting in a sum evaluation value of 4 to 25 points.
 2. Themethod of claim 1, wherein the sum evaluation value is at least 12points.
 3. The method of claim 1, wherein the porcine pancreatic isletsor the human pancreatic islet cells produce insulin; measuring theinsulin; and wherein the amount of insulin is 0.4 to 1.2 μg/l.